BY

111 IS!)Ci Syscr stated that tlic lyiiipli glatids in sliecp aid cow enibryos arise froiii a plexus of lymphatic vessels.‘ “The connectirc- tissuc between the lymphatic! vessels of the plcsns has at first a trabecular arrangement, but later one or iiiore coin1)aet masses or islands are foriiied within it. From the Legiiiiiiiig, the conncctiw tissuc which iiiakes the trabccnlae, or niasws, is ~iarrowcriiieshed than that which surrouiids it, and contaiiis niaiiy blood vcsscls.” However, he adds: “There can be 110 doubt that there are inaiiy plexiis forinations in embryonic tissue, hariiig exactly the appearance uf those from which glands arise, which simply degenerate.” Kliiig, in 1901, emphasizeil the iiuportance of the plexus stay aiid modelled the lyniphoid trabeculae.2 Although they connect with one another so as to form a continuous mass, his model has “an extremely irregular appcarancc.” It shows that these structured have little resemblance to the futnre glands. Kliiig stated that from such a general mass portions were separated by constriction to foriii the basis for individual gla~ids. Rut “Iyniph glaiids which have an isolated position appear froni the first as solitary foriliatioils ; each one arises independently.” A year later Miss Sabin wrote:” “All of the iiorlrs of the early

’Saser, F., 1’el:er die E:nt\\-ickeliiiig nnd den Bail cler iioriiialeii Lyu1p11- tlriisen. hint. EIeftc, 1WO. rol. C,, 1’1). .%O-532. Xliiig, C. A., Studien iitiar die Eiitwic.lching dpr Lyiiiplidriirzen beiin Menschen. Arch. f. inikr. Anat., 1904, vol. (3, ])[L B’iWilO. Y+abin, F. R., The developiiieiit of the lyinphatic nodes in the Iiig cult1 their relation to the lymph he‘nrts. .\mer. Joiirn. of Anat., 1905, vo1. 4, pp. ;%55-3m. (341) ttiibryos, the priiriary iiotlcs in the sense of Gulland, pass through this (plexus) stage. Lynipliatic nodes which clevelop later in the life of the t.ml)ryo, after lymphocytes occur, hurry through the primary process and show a considerable modification of it.” Recently4 I)r. Sabin published the figure of a section through the jugular lymph sac in a human embryo of 30 nim., “to show the simplc bridging of the sac which is the adage of the first lyinph node.” In thc pig she found that “the first node to appear develops from thc lymph heart, which is in the snpr a-cl av ic ular triangle be11 iiid the stcrnocleido- iriastoid muscle.” This, Saxcr, Kliiig and Miss Sabin agrw that thc first lymph glands arise from trabecnlac in a plexus of lyniphatic vessels. The plesiis of lymphatics in relatioii with thc iiitcrnal jugular veiii is a conspicuoiis featurc in human embryos iiicasuring from 30 to 40 iiiiri. Tt is showwi iii Figs. 1 ant1 2 from an embryo of 42 mm. A por- tion of the vein is sccii iii the lowcr right coriiw of each photograph. Iii places the coniiwtivc tissw trahecwlac arc broad aiiil pale, a< shown iii Fig. 1. Elsewhere they are morc slciider and deeply stain- iiig, as in the left part of Fig. 1 aiici in Fig. 2. Thc latter is a sec*tioii through the structure whicah Miss Sabin has closcribcd as the priinwy lpph gland. The cells in the similar trabeculae of a 31 mm. human embryo art! described by Kling as having “chiefly, if not exclusively, thc char-

*Sabin, 1’. R., The lyinphritic systeiii iii Iruiiiaii eiilbryos, with a con- sideration of the 111oi~111ologyof the systeiii as a wholc. Aiuer. Joiirii. of .Illat., 1909, vol. 9, py. 43-91.

. ~ ~- __ __ ESL’LANATION OF FIGS. 1-6. FIGS.1 mid 2.--I’lesus of lymphatic rerscls in reliltion with tlie jiiternal jugular rein. From a hnina1i eiiibryo of 42 inin. x 46 dinins. (Harvartl Einbryologicnl Collection, Series 841, Section 432, aiid Series 838, Sectioii 153, respectively). Fras. 3, 4, niid 5.-Lyiiipli glands froiii a huiiian embryo of 42 111111. X 60 diains. E’ig. 3 shows tlir suhiiimtcll (“subinaxillary”) gland (Series 841, Section 569) ; Fig. 4 shows tlie external jugular glaiid (Serles S41, Section 534) ; nnd Fig. 5, the circunifl1.x scapular gland (Series 838, Scctioii 321). Fro. G.--Snbscalnilar Iymldi pliiiid froiii a rribbit of 20 days, 29 iilin. X 60 diiiiiis. (11. 14:. C., Series 170, Section l0SO.)

344 Frederic T. Ikwis. acter of fixed coniiective tissue cclls.” At 70 niin. “me fiiid amoiig palc oval nuclei, others of rounder form aid darker stain which ahead) suggest adenoid tissue.” Similarly, in pigs of 80 nim. Mibs Sabin found largc, faintly staining, oval nuclei belonging to conncc- tive tissue, and small, round, cleeply staining nuclei with coarser chromatin granules and a more distinct membrane, which belong ta lyrnphocytes. “Retwecn thc connective tissue cell, especially the young forms, and the lymphocyte oiic can see every possiblc transi- tion” (1905, p. 371). Saser likewisc~found that “the lymphocyttbs, which latw forin the biilk of the lymph glaiids, arisc in loco.” The esaniination of the bridges in the 42 111111. ciii1)rpo shows the palc oval cells arid the darker round ones appareiitly dcrivd froii them, and indicates that these trabeculae contain lymphoid tiusw. They do not, however, constitute a lymph gland, hut represent thc material from which the chain of deep cervical lymph glaiids is to be derived. A suficicntly detailed study of the later stages of the plesiis has not get been made. RonliOtJ believes that it prodiicos the “inter- scapular gland” of IEatai, which seeins to bc a collective term for the cervical fat and lymph glands. Almost simultaneously with the lymphoid transformation of tra- brcirlae among the jugular lymphatics, distinct lymph glands appear in the superficial tissues. These do not pass through a plexus stage, hut from the first they resemble the glands of the adult. The striking difference in the arrangement of the deep and the superficial lymphoid tissue seems due to the fact that the deep tissue is molded about an involved pre-existing plesus ; but thr superficial glands tlerclop freely in the loosc subcutanrow tissue. The plexus stagc niay thereforc be rcgarded as a complication in the development of the glands, rather than n fundamental condition which is sometimes hurried through, modified, or omitted. In t,he human embryo of 42 mm. two superficial glands were found on either side of the head. Their position is indicatcd in Fig. 7. The smaller gland is in intimate relation with the submental branch of the anterior facial vein. A section through it is shown in Fig. :{.

&Bonnot, E.. The interscapular gland. Journ. of Anat. and Phys., 1908, vol. 43, pp. 4568. .\t thr iip1~~1)ordrr of the photograph a part of Illec*kcl'scwtilagc is .SWII oil the left, a11d the boiic of the lower jil\V oii the right ; the lower bort1t.r of the photograph passes throiigh the submaxillary glaiid. Betweeii the siibniasillarj gland aid the iiiaiitliblc the sulmieiital win appears, sn~~oinicledliy tlcnse tissue. This clciisc lyiiiphoid tissue is chiefly 011 the upper side of the veiii, aiitl it is bouiidcd by a lymphatic

FIG. 7. FIG. 8.

E'io. S.-'l'lie head of H huiiiaii eiiillryo of 42 iiiiii.. to show tlie positioii. of the suhiueiital ("subiiiiuilltir~")~iiitl esteriial jngnlar glniitls. x 3d/R iliains. (H. E. C., EM.) E'ic;. &--The liead of II ralibit eiiiliryo of 29 iiiiii. to show the position of the posterior facial glaiid. X 4 dianis. (11. E:. C., 170.) The veiiis sliowii are tlie anterior arid posterior facial, the liiiguo-facial, the external ~iidiiiteriinl jugular, iuid the j~igulo-c.e~ihalic.(The external jugular of iiiaii vorreslioiitls with tli6. jiipnlo-c.e]~litilic.of the ixbbit and iiot \villi (he li~iguo-facial; the latter iii tlie rall1)it is. howwer. ul;ually called tlie esleriial jugular. (:f. I,e\vis, Aiiier. Jonrii. of Ailat., vol. 9, 1,. 33.)

vessel, c:rescentic iii seetioil. The snbiiieiittil wi!i sends braiiches into arid throiigh the lymph gland. The other lymph gland iii tlic licad is iii relatioil with the external jugular win. It is shown iu scction in Fig. P. Lymphoid tissues, enclosing small blood vcssels, fornis a roiuided iiiass attached to thc lower part of the vein. Its free surface is in relation with a crcb- ceritic lyinph sinus. No other 1pph glands were found in the head of this embryo. :Mi Frederic T. Lewis.

In a human eiiibryo of 30 nim. the submental' aid external jiiguI;.iib glands were not found. They are not rrientioned in four embryos of 46-50 mm. described by Miss Sabin, but she has recorded that in an

FIG.9.--Itecoiistruction of tlie artcries in the axilla of the hiininn eliibryo of 42 min., to show the positioii of the first axillary lymph gland. x 10 diaim (H. P:. C., 535). The suhscapular br:uicli of the asillary artery is seen to divide into the circumfles scapular and thorwco-dorsal arteries. The lyinph gland is aloiig the latter. The brachial and latcriil thoracic arteries are also shown.

80 Imn. einbryo "therc arc scw~iidarylynipli nodcs aloiig the veins of the neck; for esaiiiple, aloug the external jugular rein next thtt parotid gland and along the facial vein at the angle of the jaw."

#It seems desirable to name the early lyniph glaiids for the veins which they accompany and this has been done. It is to be noted, however, that in the adult there are several glands along the submental vessels, the anterior ones forming the submental gronp. mid the posterior ones the submaxillary group. The submental gland of tlic 42 min. embryo belongs evidently with the submaxillary group of the adult. Lynipli Glawls in Rabbit :ind Hiunil~iEiiibrgos. 347

Sirice the early lyniph glaiids develop with such regularity in thc rabbit, it seeins quite possible that these glands noted in hnmau enibrjos of 80 min. are the ones appearing at 42 inni. The Harvard collection inclndes three rabbits of 29 mm. (90 days) cut in the traiisrersc, sagittal and frontal planes respectirely.

These enibryos all show a 1~1i~lhgland near the jnnc.tion of tht: anterior arid posterior facial wins (Pig. 8). Except at this point, no lymph glands were foiiiid iii tlic hcad. The most distinct lyriiph gl~iidin the body, in these rabbits and iii the human embryo of 42 niiii., is iii the*axillary rcgion. In th(h hiiiii;iii eiiibryo it is a11 accLuliiilatioii of 1yiiil)lioicl tissiic snrruuiiding 315 Frecleric T. Lewis. the circiuiiflcA scapular artcry and rciii, and foririing a lenticular Inass bnlgiiig into thc ac:eompaiiyiiig lyiiiphatic resskl. Its positioii is shown in Fig. 9, and a sectioii throiigh it is photographed in Piq. 5. It lies iiext the iiiuscle in the deep subcutaneous tissue. This glaiitl was not found in a 30 niiri. cnibryo, althongh at that stage thc circniiiflex scapular ressels are accompanied by lymphatirs. Tt is not specifically nieiitioned by Miss Sabin, and if it occnrrecl in the rnibrp stndied by liliiig it was overlooked. At 70 1iiiri. he found all of the axillary groiip rcprcseiitecl cxccyt the subsoapnlar group (1). ass).

&'lo. ll.-Wax recoiistriiction of the Iini~~aiiasillary glaiid shown hi Figs. 5 and I). X 40 diams. A. cir. w. 1'. vir. w.. vircuiiifiex scapular artery and rein; 2. 11. z., sii~allblood vessels, of whit-11 B is Ho surrouiideil by lymphatic vessels, 1'. 7j/t~., that it see1118 to lierforute theiu ; l).-gZ., L.-gl'., nodules of lyiiiphoid tissue.

A correspoiiding glaiitl occiirs iij rabbit cuibryos. It eaii 1)c identi- fid in a spwinicii Incasiiriug 25 iiiin. ( 18 clap), and it is wcll de- fined in all thrw of thc 2!) itiiii. eiiibryoa. Tt is in rclatiori with the sbbscapnlar vesscls, which are relatively large in the rabbit (Fig. 10A). ,4 section through the gla~idis shown in Fig. 6. Since the asillary glands seem to he the largest and most vlearly clefiiied, they wcre rcconstriirtc(1 in was. The gland iii the human eiribrp is shown in Fig. 11. 31oiig the top of the iriodcl the circnm- flex srapular artcry and veiii piirsiie u parallcl coiirse, accoinpanied by the lyrnphatic vessel*. 1'. Zyui. As the blood vessels approach the glad the mesenchyina around thcni becomes conderised and forms an intensely staining maw of lyvinphoid tissue, IAjd'. Both artery and Lyniph Glands in Ra1)l)it aii(1 TTnniaii Embryos. :340 vein are siirrounrled by this tissne, but the vein seeins more deeply embedded. The lynil~hoitltissue extends for some distance along these vessels arid forms a secoird iiodnlar swclling, L.-gl. The posi- tion of these swellings may be dcteririiiicd by thc small branches of the blood vessels, y and z, whicah they accompany. The main mass of ly~iiphoidtissne, L.-gl., foriris a leiiticnlar nodiile bulging into the pcrirasciilar lymphatic ; it has hrcn csl)nsed by removing a part of the wall of the lymphatic ressel. In the photograph, Fig. 5, the dark

FIG. l’L.--Wnx recoiistructioii of tlie asillnry glaiid of the rnbhit shown in Wigs. 6 and 70 A. >( 58 diaiiix. A. s~rhsc.,1.. subsc.. snbscnpiilar artery and vein; 1’. l!/ni., ~)~rirnsculur1yiiilihatic.s ; L. gl., 1yml)h glaiid; fr, 71, c, d, the blood vessels correrqmidingly lettered iii E’ig. 10 A. oval area is L.-p?. of the mot14 and the somewhat triangular mass aborc it is L-gl’; in the mitlst of the latter the resscl z rriay be seen. Fig. 5 is therefore a horizoiital section of thcl inodel. The fact that there are two nodular masses of lyniphoid tissiis connected with OIIC another snggcsts the twin glantls (Zwillin~sdriiser~)which Kling re- garded as Inalforinat ions duc~to iric.oiiil)lctesubdivision. The biilging of the lyinph gland I;.-gl. into the lymphatic rcssel recalls the follow- ing observation by €hivier :i “Whenever I haw observed a vascular nodule on a lymphtitic, the Itittc~has appmred to be iriterriipted. 350 Frederic T. Lewis.

. . . . Thus, the lymphatic, divided at the level of the nodule, forms two trunks, of which the inferior becomes an afferent and the superior an efferent. If a new gland forms along the course of the efferent the latter will become the afferent for the second gland. The efferent for one gland may be the afferent for another.” The subscapular gland of the rabbit is shown in the niodel, Fig. 12. The subscapular vein and artery are spun about with perivascular lymphatics, which extend along the branches of the blood vessels, a. b, c and d. (Compare with Pig. 10,4.) The lymph glaud L.-gZ. is seen through a window cut in the . It rests upon the subscapular rein and bulges into the lymphatic, pushing the endothe- lium before it. That the gland is more intimately relared to thc vein than to thc artery is shown in Fig. 6. The upper portion of the gland is irrcgnlarly snbclivided, so that in one or two sections tliere is a sug- gestion of the plexns formatioli ; Ioivor clow~iit forins a siiigle rounded inass. In addith to tlic well-defiiicd gland iii each axilla, otlicr glands were found in thc thoracic region of both thc liuinan aiid rabbit embryos. I11 the human embryo tlierc is indication of lymphoid tissnc along the dorsalis scapulae ressel aiitl a somewhat diffuse gland iiear the anterior end of the interrial inuminary vein. Whcro the pleuro- pericardial scptum joiiis the diaphragm a brarwh of the iriteriial mammary vciri passes inward, acconipaiiicd by a largc lymphatic. Near the junction of the septum aiid diaphragm lymphoid tissue is found in relation with these vessels. The left pleuro-pericardial scptum is thinner and farther from the median liiie than the right and has no corresponding lymphoid tissue. In the rabbit there is a devel- oping gland along the thoraco-epigastric, or external mammary vein, nearly opposite the elbow. The glands of the head and thorax have now been described ; the abdominal and pelvic wgions remain to be considered. In four rabbit embryos of 20 nim. a gland was found along the ilio-lumbar vein on either side of the body. Tt appears to be smaller than the axillary gland, but has essentially the same features. It is more deeply placed than the other glands. Thc ilio-lumbar vessels (Fig. 1011) have exten- sive subcutaneous branches, f, f, f, and a branch, e, to the abdomind Lymph Glands in Rabbit and Human Embryos. 351 musculature. As noted by Krausr, the ilio-lumbar vessels are highly developed in the rabbit. The lymph glaiid is found, as shown in Fig. 10B, where the subcutaneous branchcs joiii the main stem. In the abdominal part of the human ernbryo of 42 mm. no distinct glands were found, but along the femoral vessels, in the iuguinal region, there is a suggestion of lymphoid tissue. At 60 mm. Miss Sabin describes the posterior lyiiipli sacs as lying in the side of the pelvis opposite the first three sacral vertel)rae, rtlid states that “the entire dorsal wall of the sac is occupied by a lymph node” (190!), 1). 87). ,4 gland which extcnds over three sacral vertebrae is clearly unlike any gland in the adult. The striwture referred to seeins to he the plexus of deep lymphatics, aiiiong whivh lymphoid tissue has np- pearrd, but has not yet formed glaiids. At this stage Miss Sabin speaks of “sccoiidary nodes” developing iicar the sacs along the femoral and sciatic gronps of vciiis. In a11 YO iitiii. ~Iii~J~~Oshe describes a true lymph gland wliich, from its stritctarc aiid I)ositioii, as shown in a figure, strikingly snggchth thc ilio-lmnbar glaiicl of the rabbit; it is iiot stated aloiig what vessels it owurs. The description of the gland is as follows: “Ill Fig. 19 is a tiny lymph node . . . which illustratcs well the siiiiplest form of a l~mphnode, a central mass of Iyniphocytes with a plexus of lymph dncts around it. This plexus of dncts is so closc that it may already be tcriiied a siniis, so that the node coneists of a siiigle follicle with its periphcral siniis.” Tt may be noted that Miss Sabin has fignrt.tl siwh u simple gland in the Iting of an adult pig (19OS, p. R%), and Rling has described them in the axilla of an adult man. From the preceding study the coriclusioii may be drawn that the first definite lymph glands are suprrficial. They appear with siir- prising regularity, as shown by comparing the three rabbit embryos of 29 nun. They are situated along the large cutaneous veins, arid there is a well-developed pair for the head, thorax, and abdomen re- spectively. In addition to these, the rabbit embryo of 29 mm. gives evidence of gland formation along the thoraco-epigastric vein. The. human embryo of 42 mm. differs from the rabbit, of 20 mm. by tile absence of the ilio-lambar gland and the presence of the submental gland, togethrr with inclicatioiis of glands along thr internal main- 352 Frederic, T. Lewis. inary and femoral veins. Doiibtless, both in man aud the rabbit the development of additional glands procceds rapidly. At the time whcn the superficial glancls are distinct the deep ones are represented by lymphoid trabc~dae,which are said to be trans- formed into chains of glands by the accumulation of the lymphoid tissue in nodnlrs. Soiriethiiig of this sort must occiir, but models slowing the developmciit of such a chain have nvt yvt been made. It seems iuidesirable to speak of a11 extensire plexus of lymphatic vcs- scls, even ~vhenassociated with diffuse lymphoid tissue, as a lymph glad. At the time whcn the lymph glands arid t,rabeculae arise-that is, in the embryos which have now been described-there is apparentlj nu lyniphoid tissue elsewhere in the hly. The is well devel- oped, hit the compact tissiie of which it is composcd does not appear like that of the lymph glancls. The thyrniis at this stage, in the rabbit at least, is clearly an epithclial . This is contrary to the state- irient of Gulland,8 that “thp tlijiiiiis in inarninaliaii einhryos is the first place where true ahloid tissiie is formed, and it is a11 active e~iitcrfor the production of leucocytes long before lyinphatic glaiirls are formed at all.” The question of the origin of Iy~nphocytescan be answered oiily by examinifig thin and .specially stained sections. The embrFos here described were prepared for gcneral study, and the sections are 10 microns or 1noi-e in thicknrss. They Siiggpt,, howevcr, that the Jyniph- ocaytes are forming in the glands and that thry are absciit from the blood. Maximow, who has stntlied the einbryonic tlcvelopnient of the blood with faiiltless technique, tias nnfortiinatcly not oxamiried die carliest lymph glands.9 He consiclrrs that “the first leucocytes, the lymphocytes, arisr at thc same hie and from the same soiirce as the primitive erytliroblasts. Thc latter 1.c prewnt a specially differentinted form of cell, but the lymphocytes always remain iuidifferent iattd.

‘Gnllund, G. I,., The developinelit of lymphatic glantls. Joiirn. of Path. and Boct., 18M, vol. 2, pp. 447-485. Warinow. A., L‘ntersuchung uber Blut uiid Uiudegewebe. I. Die frilhesteri Entwicklungsstadien der Blut. etc. Arch. f. aiik. Anot.. 1909. vol 73. pp. 444-561. Lymph Glands in Rabbit and Human Embrjos. 353

Therefore, like tlie p-itiiitive blood cells froiii which they directly proceed, they are uridifferoiitiated rounded amoeboid mesenchymal cells.” He states that these lymphocytes of the embryo “have nothing to do with lymphoid tissue”-they derelop in the yolk sac. The 1ymphocTtes of the lymph glands are, indeed, round mesen- chynial cells, but, except for an occasional cell in the lymph sinus, apparently detached from the gland, they are unlike the forms of corpuscles in the adjacent vessels. It seeins probable that the lyinph glands, arising in rabbit embryos of 25-30 mm. and in huinaii embryos of 30-45 mm., are the source of a special form of round inesenchymal cell, which is the true lymphocyte. This opinion can be established or disproved only by a cytological study of thc early lymph glands, the position of which has been indicated.